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Minnesota/22 July 2008
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|'''4. Vector Dephosphorylation:''' Same dephos. procedure used on GFP:Terminator, BV. After dephosphorylation, incubate @37C for 30 mins. Heat inactivate dephosphorylation enzyme for 15 mins in 65C water bath. | |'''4. Vector Dephosphorylation:''' Same dephos. procedure used on GFP:Terminator, BV. After dephosphorylation, incubate @37C for 30 mins. Heat inactivate dephosphorylation enzyme for 15 mins in 65C water bath. | ||
|- | |- | ||
| - | |'''5. Ligation Reactions:''' | + | |'''5. Ligation Reactions:''' Procedure performed in 20 minutes. Once all were ligated, were then incubated @ 16C for 1 hour. Heat inactivated enzyme @ 65C for 15 minutes. Ligated the following using L4 DNA Ligase: |
| + | |- | ||
| + | |'''a.''' BV + TetR:p22 promoter + RFP | ||
| + | |- | ||
| + | |'''b.''' BV + TetR:p22 promoter + YFP | ||
| + | |- | ||
| + | |'''c.''' LacI:LambdacI + GFP:Terminator | ||
| + | |- | ||
| + | |'''d.''' LacI Promoter + p22 cII + BV | ||
| + | |- | ||
| + | | | ||
Revision as of 20:55, 21 July 2008
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| 1. Pick Colonies from plates made 07-21-2008 Start cultures. |
| 2. Plasmid prep: Prep RFP, YFP, GFP, TetR promoter, Terminator. Follow QIA miniprep procedure --> 1hr long. |
| 3. Double digest: Follow Kat's DNA work procedure to perform Double digest on: LacI Promoter, p22 cII gene, RFP, YFP, BV (dual promoters, GFP:Term already d.dig.). Incubate digested products for 2 hours @37C. Heat inactivate digestion enzyme for 15 mins @ 65C water bath. |
| 4. Vector Dephosphorylation: Same dephos. procedure used on GFP:Terminator, BV. After dephosphorylation, incubate @37C for 30 mins. Heat inactivate dephosphorylation enzyme for 15 mins in 65C water bath. |
| 5. Ligation Reactions: Procedure performed in 20 minutes. Once all were ligated, were then incubated @ 16C for 1 hour. Heat inactivated enzyme @ 65C for 15 minutes. Ligated the following using L4 DNA Ligase: |
| a. BV + TetR:p22 promoter + RFP |
| b. BV + TetR:p22 promoter + YFP |
| c. LacI:LambdacI + GFP:Terminator |
| d. LacI Promoter + p22 cII + BV |
